CN106661602A

CN106661602A - Chemically-locked bispecific antibodies

Info

Publication number: CN106661602A
Application number: CN201580033933.3A
Authority: CN
Inventors: Y·福; G·F·考夫曼; B·琼斯; R·图赫里
Original assignee: Sorrento Therapeutics Inc
Current assignee: Sorrento Therapeutics Inc
Priority date: 2014-05-10
Filing date: 2015-05-10
Publication date: 2017-05-10
Anticipated expiration: 2035-05-10
Also published as: US20170260291A1; AR100367A1; EP3143154A1; CA2948810A1; KR102460736B1; JP2017518975A; EP3143154B1; KR20170002622A; AU2015259533B2; CN106661602B; WO2015175357A9; EP3143154A4; AU2015259533A1; US10435479B2; JP6687247B2; WO2015175357A1

Abstract

There is disclosed a process for forming chemically-locked bispecific or heterodimer antibodies, preferably in the IgG class, in high specificity and with high homogeneity. More specifically, there is disclosed a chemically-locked bispecific IgG class antibody having a linkage region joined together with bio-orthogonal click chemistry.

Description

The bispecific antibody of chemistry locking

Technical field

Present disclose provides the bispecific or heterodimeric for forming chemistry locking with high specific and high homogeneity The method of body antibody (preferred IgG classes).More specifically, utilizing bio-orthogonal click chemistry (bio- present disclose provides having Orthogonal click chemistry) connection bonding pad chemistry locking bispecific IgG antibody-likes.

Cross-Reference to Related Applications

Patent application claims on May 10th, 2014 submit to No. 61/991,508 U.S. Provisional Patent Application it is preferential Power.

Background technology

Immunoglobulin G or IgG antibody exist with four subclass, and each subclass has different 26S Proteasome Structure and Functions special Property.IgG is made up of two heavy chain-light chains to (incomplete antibody), and the heavy chain-light chain is residual to the Cys in by being directly connected to hinge area Base (EU index numbers：Cysteine residues 226 and 229；Kabat is numbered：Two between cysteine residues 239 and heavy chain 242) Sulfide linkage connects.The molecule of human IgG 4 with molecular species presence, its by weight interchain disulfide bond presence or absence of and it is different.

Diversified recombinant antibodies form is had been developed for, such as by merging IgG antibody form and single domain The tetravalence bispecific antibody (159-163 of Coloman etc., Nature Biotech 15 (1997)；WO 2001/077342；With The 1233-1234 of Morrison, Nature Biotech 25 (2007)).Another kind of form has the antibody core knot for no longer retaining Structure (IgA, IgD, IgE, IgG or IgM), such as Diabody, three body antibody or four body antibody, miniantibody, several single stranded forms (scFv、Bis-scFv).But this kind of form can be with reference to two or more antigens (Holliger etc., Nature Biotech 23(2005)1126-1136；The Fischer and 3-14 of Leger, Pathobiology 74 (2007)；Shen etc., J.Immunological Methods 318(2007)65-74；With Wu etc., Nature Biotech.25 (2007) 1290- 1297)。

Report in US 2005/0163782 for from comprising wherein by the dimerization of at least one interchain disulfide bond connection Body and wherein by least one interchain disulfide bond connection the dimeric two types mixture in will be by least one The dimer of individual interchain disulfide bond connection is separated with the dimer not connected by least one interchain disulfide bond or preferentially synthesized The dimeric method connected by least one interchain disulfide bond.

Bispecific antibody is to be difficult with traditional hybrids knurl and chemical conjugation methods with sufficient amount and quality The material of generation.Additionally, WO2005/062916 and U.S. Patent application 2010/0105874 are describeed how by going back original antibody " AA " and antibody " BB " with by disulfide bond be separated into the chain of the substance with single calmodulin binding domain CaM-light chain unit (A or B) (wherein A and Both B are with reference to different targets) forming bispecific antibody.Then, antibody allows disulfide bond that isomerization occurs, so that anti- Body AB, BA, AA and BB are each recombinated with about 25% probability.However, both AB and BA are identical bispecific antibodies, and because This at most shows about 50% yield.Therefore, this need extra step by the bispecific antibody needed for being formed from Original recombinant antibodies are separated.However, U.S. Patent application 2010/0105874 points to the hinge in the IgG4 with CPSC sequences Sequence, and state：" CPSC sequences cause more flexible core hinge and form the possibility ... of intrachain disulfide bond. it is believed that tool The antibody for having IgG4 sample core hinge sequences can have the intrinsic activity for resetting disulfide bond, and it passes through in the inventive method The condition simulation for using " (the 0013rd section).Additionally, being prepared for the bispecific antibody of other forms, it has by changing Become " pestle and mortar " structure of the sequence of heavy chain generation of antibody A and B.

Therefore, present disclose provides the method for producing the bispecific IgG antibody of chemistry locking, it meets this area to double The pestle and mortar method of the amino acid sequence in the needs and the antibody regions more fixed than change of the very high yield of specific antibody With more preferable stability.

The content of the invention

Present disclose provides be used for from IgG1, IgG2 or IgG4 antibody-like or its Fab2 fragment " A " and IgG1, IgG2 or The method that IgG4 antibody-likes or its Fab2 fragment " B " produce the bispecific antibody " AB " or " BA " of chemistry locking.The method bag Include：

A () makes the first antibody under conditions of it be enough to cut the essentially all disulfide bond in hinge area between heavy chain " A " is contacted with reducing agent, and to produce a pair of first antibody fragments A', each first antibody fragment A' is included and is connected to single heavy chain Single light chain, wherein the heavy chain has one or more reactive sulfydryls for being formed by the reduction of the disulfide bond；

B first Heterobifunctional joint is connected to first antibody fragment A' by (), wherein first Heterobifunctional connects The first sulfydryl reaction that head is used to be covalently attached with the reactive sulfydryl of the heavy chain of first antibody fragment A' comprising (i) Property functional group, and (ii) azide, so as to form the first antibody fragment of azide functionalization；

C () makes the SA under conditions of it be enough to cut the essentially all disulfide bond in hinge area between heavy chain " B " is contacted with reducing agent, and to produce a pair of SA fragments B', each SA fragment B' is included and is connected to single heavy chain Single light chain, the heavy chain has one or more reactive sulfydryls for being formed by the reduction of the disulfide bond；

D second Heterobifunctional joint is connected to SA fragment B', the second Heterobifunctional joint bag by () Contain：I () is used for the second sulfydryl reactivity official being covalently attached with the reactive sulfydryl of the heavy chain of the SA fragment Can roll into a ball, and (ii) alkynes；So as to form the SA fragment of alkynes functionalization；

E () makes the first antibody fragment of the azide functionalization react with the SA fragment of the alkynes functionalization So that the first antibody fragment is covalently attached into described with the 1,3- dipole-diople interactions of the alkynes by the azide Two antibody fragments, so as to form the bispecific antibody " AB " or " BA " of chemistry locking.

Preferably in the bar for substantially not reducing the disulfide bond between heavy chain and light chain the step of disulfide bond in reduction hinge area Carry out under part, it means that, in some embodiments, at least about 90% or at least about 95% or at least about 99% it is this kind of heavy Disulfide bond between chain and light chain keeps complete after the disulfide bond cracking in hinge area.

Preferably, the first Heterobifunctional joint has Q-L-N₃Form, wherein Q is sulfydryl reactive functional groups, and L is hydrocarbon Joint, and N₃It is azide.Preferably, sulfydryl reactive functional groups Q is alkyl halide (for example, alkyl chloride, alkyl bromide or alkane Base iodine), benzyl halide, maleimide, halo maleic amide (bromo maleimide) or dihalo maleimide (two bromos Maleimide).Preferably, L is in Q and N₃Between there is in straight chain 3-60 atom (for example, being more typically 6-50 individual) Hydrocarbon joint.It is highly preferred that L is polyalkylene oxide (PEF) group or L is that wherein each chain link (mer) unit is-(CH₂CH₂–O)_n Or (O-CH₂CH₂)_n- polymer (wherein " n " is independently the integer of 1-20, more typically 1-8).

Preferably, the first isodigeranyl functional connector (being connected to first antibody fragment) is：

Wherein Q can be suitable for any group being connected to joint on antibody fragment, but be preferably able to conducting oneself with dignity The sulfydryl covalent bonding of the cysteine residues in chain hinge area.Exemplary groups Q is：

Wherein Z is independently selected from H, Br, I and SPh.Preferably, at least one Z is not H, although in the feelings of maleimide Under condition, Z can be hydrogen when occurring every time.M independently is CR^*Or N.

X₁、X₂、X₃、X₂、X₄And X₅Independently selected from key (not existing) ,-O-,-NR^N- ,-N=C-,-C=N-,-N= N-,-CR*=CR*-(cis or trans) ,-C ≡ C-,-(C=O)-,-(C=O)-O-,-(C=O)-NR^N- ,-(C=O)- (CH₂)_n-, (C=O)-O-(CH₂)_n-, (C=O)-NR^N–(CH₂)_n- and-(C=O)-NR^N–(CH₂CH₂–O)_n-, wherein " n " is 0 Or the integer of 1-10.

R^a、R^b、R^cAnd R^dIndependently selected from-O-,-NR^N–、–CH₂–、–(CH₂)_n–、–(CR*₂)_n–、–(CH₂CH₂–O)_n–、– (CR*₂CR*₂–O)_n–、–(O–CH₂CH₂)_n–、–(O–CR*₂CR*₂)_n- ,-CR*=CR*-(cis or trans) ,-N=C-,-C= N-,-N=N-,-C ≡ C-,-(C=O)-,-(CH₂)_n- (C=O)-,-(C=O)-(CH₂)_n–、–(CH₂)_n- (C=O)- (CH₂)_n- ,-O-(C=O)-,-(C=O)-O-,-O-(C=O)-O-,-(CH₂)_n- (C=O)-O-,-O-(C=O)-(CH₂)_n、– (C=O)-O-(CH₂)_n–、–(CH₂)_n- O-(C=O)-,-(CH₂)_n- (C=O)-O-(CH₂)_n–、–(CH₂)_n- O-(C=O)- (CH₂)_n–、–NR^N- (C=O)-,-(C=O)-NR^N–、–NR^N- (C=O)-O-,-O-(C=O)-NR^N–、–NR^N- (C=O)- NR^N–、–(CH₂)_n- (C=O)-NR^N–、–NR^N- (C=O)-(CH₂)_n,-(C=O)-NR^N–(CH₂)_n–、–(CH₂)_n–NR^N- (C= O)–、–(CH₂)_n- (C=O)-NR^N––(CH₂)_n–、–(CH₂)_n–NR^N- (C=O)-(CH₂)_n- ,-(C=O)-NR^N–(CH₂CH₂–O )_n–、–(CH₂CH₂–O)_n- (C=O)-NR^N–、–(CH₂)_n- (C=O)-NR^N–(CH₂CH₂–O)_n–、–(CH₂CH₂–O)_n- (C=O)- NR^N–(CH₂)_n- and 2-8 units cyclic hydrocarbon, heterocycle, aryl or heteroaryl ring；Wherein " n " independently is 0 or 1-10 integer；With Wherein " l ", " p ", " q " and " r " independently is 0 or 1-10 integer.

Ω is key (that is, it is not present) or C_3-26Hydrocarbon ring or carbocyclic fused ring system, optionally comprising at most four fused rings, Wherein each ring has 3-8 units and optionally in each ring comprising the 1-4 hetero atom selected from O, S and N.Preferably, Ω is The 1,2,3- triazole rings for condensing with cyclooctane ring or condensing with 8 circle heterocycles or loop systems.

R^*And R^NH or C independently is when occurring every time_1-12Hydrocarbon, optionally by 1-6 selected from halogen, the hetero atom of O, S and N Replace；With any two of which group R^*And/or R^N3-8 yuan of rings can together be formed.

The second Heterobifunctional joint has the form of Q-L-G, and wherein Q is sulfydryl reactive functional groups, and L is that hydrocarbon connects Head, and G is containing ethynylene group.Sulfydryl reactive functional groups Q is selected from alkyl halide (such as alkyl chloride, alkyl bromide or alkyl iodide), benzyl Halogen, maleimide, halo maleic amide (such as bromo maleimide) and dihalo maleimide (e.g., two bromo horse Carry out acid imide).L is the hydrocarbon joint in straight chain between Q and G with 3-60 atom (for example, it is preferable to 6-50).Preferably, L It is polyalkylene oxide groups (PEG), it is preferable that L is unit-(CH₂CH₂–O)_n- or-(O-CH₂CH₂)_n- (wherein " n " independently is The integer of 1-20, more typically 1-8) polymer.

G is that any containing ethynylene group of cycloaddition can occur with azide.In some embodiments, G includes end Alkynes, such as-C ≡ CH.In other embodiments, G is included in ring the ring with-C ≡ C- keys or loop systems.In a reality In applying mode, G includes the C8 rings with-C ≡ C- keys.In one embodiment, the ring containing-C ≡ C- is strain (strained)。

Second heterobifunctional linker has following form：

It is identical or different in Q and the first heterobifunctional linker.Q is typically the sulfhydryl reactive group of following form：

Wherein Z is when occurring every time independently selected from H, Br, I and SPh.In some embodiments, at least one Z is not H, although in the case of maleimide, Z can be hydrogen when occurring every time.M independently is CR when occurring every time^*Or N.

G is typically comprising the C of-C ≡ C- keys that 1,3 Dipolar Cycloadditions can occur with azide_8-20Alkyl. In some embodiments, G has the form of-C ≡ C-H.In other embodiments, G includes the ring with-C ≡ C- keys.Especially Ground, G can include the 8- yuan of rings (such as cyclooctyne) with three keys.The ring optionally with one, it is two or more in addition Ring condense, the other ring is usually C_3-6Cyclic hydrocarbon ring, including aryl, heteroaryl and heterocycle.8- yuan of rings containing three keys can be Ring includes one or more (such as 1-4) hetero atoms, such as nitrogen and oxygen.In one embodiment, 8 yuan of rings contain in ring There is nitrogen-atoms, it provides the tie point with L, such as in example arrangement shown below：

Preferably, G has following form：

X₁、X₂、X₃、X₂、X₄And X₅When occurring every time independently selected from key (that is, not existing) ,-O-,-NR^N- ,-N= C-,-C=N-,-N=N-,-CR*=CR*-(cis or trans) ,-C ≡ C-,-(C=O)-,-(C=O)-O-,-(C=O)- NR^N–、–NR^N- (C=O)-,-NR^N- (C=O)-O-,-(C=O)-(CH₂)_n- ,-(C=O)-O-(CH₂)_n- ,-(C=O)-NR^N– (CH₂)_n- and-(C=O)-NR^N–(CH₂CH₂–O)_n-, wherein " n " is 0 or the integer of 1-10；

R^a、R^b、R^cAnd R^dWhen occurring every time independently selected from-O-,-NR^N–、–CH₂–、–(CH₂)_n–、–(CR*₂)_n–、– (CH₂CH₂–O)_n–、–(CR*₂CR*₂–O)_n–、–(O–CH₂CH₂)_n–、–(O–CR*₂CR*₂)_n- ,-CR*=CR*-(cis or anti- Formula) ,-N=C-,-C=N-,-N=N-,-C ≡ C-,-(C=O)-,-(CH₂)_n- (C=O)-,-(C=O)-(CH₂)_n–、– (CH₂)_n- (C=O)-(CH₂)_n- ,-O-(C=O)-,-(C=O)-O-,-O-(C=O)-O-,-(CH₂)_n- (C=O)-O-,-O- (C=O)-(CH₂)_n,-(C=O)-O-(CH₂)_n–、–(CH₂)_n- O-(C=O)-,-(CH₂)_n- (C=O)-O-(CH₂)_n–、– (CH₂)_n- O-(C=O)-(CH₂)_n–、–NR^N- (C=O)-,-(C=O)-NR^N–、–NR^N- (C=O)-O-,-O-(C=O)- NR^N–、–NR^N- (C=O)-NR^N–、–(CH₂)_n- (C=O)-NR^N–、–NR^N- (C=O)-(CH₂)_n,-(C=O)-NR^N– (CH₂)_n–、–(CH₂)_n–NR^N- (C=O)-,-(CH₂)_n- (C=O)-NR^N––(CH₂)_n–、–(CH₂)_n–NR^N- (C=O)- (CH₂)_n- ,-(C=O)-NR^N–(CH₂CH₂–O)_n–、–(CH₂CH₂–O)_n- (C=O)-NR^N–、–(CH₂)_n- (C=O)-NR^N– (CH₂CH₂–O)_n–、–(CH₂CH₂–O)_n- (C=O)-NR^N–(CH₂)_n- or 2-8 units cyclic hydrocarbon, heterocycle, aryl or heteroaryl ring； Wherein " n " independently is 0 or 1-10 integer when occurring every time；Wherein " l ", " p ", " q " and " r " independently is 0 or 1- 10 integer；

Ω is key (that is, not existing) or for C_3-26Hydrocarbon ring or carbocyclic fused ring system, optionally comprising at most four fused rings, often Individual ring has 3-8 units and in each ring optionally comprising the 1-4 hetero atom independently selected from O, S and N；In an embodiment party In formula, Ω will include the 1,2,3-triazoles ring for condensing with cyclooctane ring or condensing with 8 circle heterocycles or loop systems.

R^*And R^NH or C independently is when occurring every time_1-12Hydrocarbon, optionally by 1-6 selected from halogen, the miscellaneous original of O, S and N Son replaces；And two of which group R^*And/or R^N3-8 yuan of rings can together be formed.

Cycloaddition reaction between azide and alkynes can be carried out by 1,3 Dipolar Cycloadditions.The reaction can be with It is catalyzed by copper ion.In some embodiments, cycloaddition reaction occurs under neutral or physiological pH.

The disclosure additionally provides a kind of for having hinge residues sequence (EU index numbers：Residue 226-229；Kabat is compiled Number：Residue 239-242) CPPC or CPSC or SPPC or SPSC goes back original antibody " A " and with hinge residues sequence (residue 226- 229) method of the SA " B " of CPPC or CPSC or SPPC or SPSC to form incomplete antibody A and incomplete antibody B, the method bag Include：

A () reduces each antibody A and antibody B, thus reducing condition destruction has hinge residues sequence (EU index numbers： Residue 226-229；Kabat is numbered：Residue 239-242) CPPC or CPSC or SPPC or SPSC each antibody hinge region in appoint What interchain or intrachain disulfide bond；

B () will be connected to one or two Cys residue of the hinge core sequence of incomplete antibody A selected from following compound (EU index numbers：Residue 226 and 229；Kabat is numbered：Residue 239 and 242) forming the incomplete antibody A of connection：

M=N, C

M '=N, C

Z=I, Br, SPh,

Wherein N₃It is-N=N=N；

C () will be connected to one or two Cys residue 226 of the hinge core sequence of antibody B selected from following compound With 229 (EU index numbers：Residue 226 and 229；Kabat is numbered：Residue 239 and 242) forming the antibody B of connection：

M=N, C

M '=N, C

Z=I, Br, SPh；With

D () incubates in neutral conditions antibody A and the antibody B being connected of the connection of approximately equal mole to form connection Bispecific antibody AB.

Preferably, antibody A reduction to form incomplete antibody B is carried out in reducing agent to form incomplete antibody A and antibody B reduction , wherein the reducing agent is selected from Cys, dithiothreitol (DTT), beta -mercaptoethanol, cysteamine, TCEP (three (2- carboxylic second Base) phosphine), 2-MEA (2-MEA) and combinations thereof.Preferably, the hinge area of the antibody A with one or two Cys residue It is connected with having the part A selected from following structure：

M=N, C

M '=N, C

Z=I, Br, SPh,

Wherein N₃It is-N=N=N.Preferably, the hinge area of the antibody B with one or two Cys residue is selected with having Connect from the part B of following structure：

M=N, C

M '=N, C

Z=I, Br, SPh,

To form the incomplete antibody B of connection.

The disclosure additionally provides a kind of bispecific antibody AB of chemistry locking, wherein the incomplete antibody A for connecting

Wherein N₃It is-N=N=N,

It is incorporated into the antibody B of connection

To form the bispecific antibody AB with structure shown in Figure 10.

Present disclose provides from IgG antibody-likes " A " or its fragment and IgG antibody-likes " B " chemistry locking it is double special Property antibody " AB " or " BA ", its include with selected from following structure incomplete antibody A：

M=N, C

M '=N, C

Z=I, Br, SPh,

Wherein N₃It is-N=N=N；Wherein Z is leaving group；

With with selected from following structure incomplete antibody B：

M=N, C

M '=N, C

Z=I, Br, SPh.

The disclosure further provides bispecific antibody, and it is included：

(a) first antibody fragment A', it includes the single heavy chain and light chain from antibody A, the heavy chain have one or Multiple reactive mercapto groups；

B () SA fragment B', it includes single heavy chain and light chain, and the heavy chain has one or more reactive mercaptos Base group；

Wherein described first and second antibody fragment formed by cycloaddition reaction via azide and alkynes 1,2, 3- triazoles are covalently attached, and the azide is connected to the reactive sulfydryl of the first antibody fragment by joint, and alkynes leads to Cross the reactive sulfydryl that joint is connected to the SA fragment.Described above is joint.Antibody fragment A' and B' derived from IgG1, IgG2 or IgG4 immunoglobulin (Ig) or its Fab2 fragment.

The disclosure further provides the antibody fragment with joint covalent bonding, wherein the joint is comprising have can be with There is the C8 rings of-C ≡ C- keys of cycloaddition reaction in azide.The disclosure additionally provides the antibody piece with joint covalent bonding Section, wherein the joint includes the azide that cycloaddition reaction can occur with-C ≡ C- keys.

Preferably, antibody A reduction is carried out with forming incomplete antibody B with forming incomplete antibody A and antibody B reduction in reducing agent, Wherein described reducing agent is selected from Cys, dithiothreitol (DTT), beta -mercaptoethanol, cysteamine, TCEP (three (2- carboxyethyls) Phosphine), 2-MEA (2-MEA) and combinations thereof.Preferably, the hinge area of the antibody A with one or two Cys residue and tool There is the part A selected from following structure to connect：

M=N, C

M '=N, C

Z=I, Br, SPh,

Wherein N₃It is-N=N=N

It is connected to one or two Cys residues (EU index number of the hinge core sequence of incomplete antibody A：The He of residue 226 229；Kabat is numbered：Residue 239 and 242) forming the incomplete antibody A of connection；

M=N, C

M '=N, C

Z=I, Br, SPh；With

M=N, C

M '=N, C

Z=I, Br, SPh

M=N, C

M '=N, C

Z=I, Br, SPh,

To form the incomplete antibody B of connection.

Wherein N₃It is-N=N=N；

It is incorporated into the antibody B of connection

To form the bispecific antibody AB with structure shown in Figure 10.

Present disclose provides the bispecific antibody of the chemistry locking from IgG antibody-likes " A " and IgG antibody-likes " B " " AB " or " BA ", it is included with the incomplete antibody A selected from following structure：

M=N, C

M '=N, C

Z=I, Br, SPh,

Wherein N₃It is-N=N=N, and wherein Z is leaving group, it is incorporated into；

With with selected from following structure incomplete antibody B：

M=N, C

M '=N, C

Z=I, Br, SPh.

Description of the drawings

Fig. 1 show arrange by with the hinge area of IgG antibody-likes in single Cys residues chemical coupling it is double to produce The schematic diagram of specific mAb.

Fig. 2 show according to the disclosure by with the hinge area of IgG antibody-likes in single Cys residues chemical coupling chain Between be crosslinked schematic diagram.

Fig. 3 shows the internally crosslinked schematic diagram of two Cys residue chains in the hinge area with IgG antibody-likes.

Fig. 4 show (upper and lower) by with the hinge area of IgG antibody-likes in two Cys residues interchain linkage produce it is double The schematic diagram of specific mAb.

Fig. 5 shows the SDS PAGE analyses of half mAb fragments of chemistry locking.

Fig. 6 show from naked mAb (on), azide be coupled mAb fragments (centre) and alkynes coupling mAb fragments (under) HC Fab MS analysis.

Fig. 7 shows the SDS of the cross-linking products of the half mAb fragments of half mAb and alkynes connection from azide connection PAGE。

Fig. 8 show from initial mAb (on) and cross-linking products (under) (Fab)₂MS analysis.

Fig. 9 shows the non-reduced SDS PAGE (swimming lanes of the incomplete antibody fragment of the chemical modification produced in embodiment hereof 4 2 and 3).

Figure 10 to show and be coupled (Click conjugation) generation pair specifically by the click between two incomplete antibody fragments Property antibody.

Figure 11 shows incomplete antibody fragment (a) and clicks on the non-reduced SDS PAGE of product (b).Incomplete antibody-azide exists In the swimming lane 2 of gel (a), and swimming lanes 3 of the incomplete antibody-DBCO in gel (a).Click on swimming lane 2 of the product in gel (b) In.

Figure 12 shows the mass spectrum of the click product of the IdeS digestion of the quality for illustrating bispecific antibody CBA-0710.

Figure 13 shows the SEC size exclusion chromatographies of bispecific antibody CBA-0710.

Figure 14 shows combinations of the bispecific antibody CBA-0710 on BIAcore.More specifically, Figure 14 shows double special Property antibody CBA-0710 and BIAcore on 2 of two kinds of antigen combinations simultaneously.

Figure 15 shows that bispecific antibody CBA-0710 is combined with triple negative breast cancer cell MDA-MB-231.The cell table Up to antigen -1 and antigen -2.STI-A0607 is the monoclonal antibody of antigen -1, and STI-A1010 is that the monoclonal of antigen -2 resists Body.

Figure 16 shows antagonistic activities of the bispecific antibody CBA-0710 in triple negative breast cancer cell.STI-A0607 It is the monoclonal antibody of antigen -1, and STI-A1010 is the monoclonal antibody of antigen 2.HGF is the native ligand of antigen -1.

Figure 17 shows that the IFN-γ release in response to bispecific antibody CBA-0710 increases.STI-A1010 is anti- Former -2 (immunologic test point) monoclonal antibodies.Competition mAb is humanization antigen -2 (immunologic test point) monoclonal antibody.

Figure 18 shows that the IL-2 releases in response to bispecific antibody CBA-0710 increase.STI-A1010 is antigen -2 (immunologic test point) monoclonal antibody.Competition mAb is humanization antigen -2 (immunologic test point) monoclonal antibody.

Figure 19 shows and compared with anti-PD-L1IgG1 antibody with conventional anti-c-Met IgG1, resisted by the anti-C-met antibodies of A and B Effect that the chemistry locking bispecific antibody of PD-L1 antibody composition improves.

Figure 20 to show and produce bispecific F (ab) ' by chemical coupling₂Schematic diagram.

Figure 21 shows the SDS_PAGE gel analysis of F (ab) ' 2 and F (ab) '.1st row are the antibody As digested with IdeS.The 2 row are the antibody B digested with IdeS.3rd row are that Jing digests and remove the antibody A of FC.4th row are that Jing digests and remove the anti-of FC Body B.5th row be that Jing digests and remove FC, with 2MEA and TCEP reduction and with DBCO (dibenzo cyclooctyl)-maleimide idol The antibody A of connection.6th row be that Jing digests and remove FC, with 2MEA and TCEP reduction and with azide-maleimide amine coupling Antibody B.

Figure 22 is shown and be coupled that producing F (ab) ' 2 chemistry locking bispecific resists by the click between two F (ab) ' fragments The schematic diagram of body.

Figure 23 shows the SEC from click F (ab) ' 2 of embodiment hereof 7.

Figure 24 shows and clicks on bispecific F (ab) '₂Fragment analysis SDS PAGE.1st row are that Jing digests and remove the anti-of FC Body A, and reduced with the 2MEA and TCEP with DBCO (dibenzo cyclooctyl)-maleimide amine coupling.2nd row be Jing digestion and The antibody B of FC is removed, and is reduced with the 2MEA and TCEP with azide-maleimide amine coupling.3rd row are click on _ bis- spies Different in nature F (ab) '₂。

Figure 25 shows the mass spectrum for clicking on product, it illustrates bispecific F (ab) '₂Quality.

Figure 26 shows the SEC for clicking on F (ab) ' 2.

Figure 27 shows bispecific F (ab) '₂Combined while with two kinds of antigen on Octet Red.

Figure 28 shows the schematic diagram that bispecific IgG2 is produced by chemical coupling.

Figure 29 shows IgG2 fragment analysis SDS PAGE.1st row are antibody As.2nd row are with TCEP reduction and and Azide The antibody A of thing-maleimide amine coupling.3rd row are antibody B.4th row be with TCEP reduction and with azide-maleimide The antibody B of amine coupling.

Figure 30 A-C show mass spectrogram, it illustrates with joint be coupled IgG2_A (Figure 30 A) and joint be coupled The quality of the bispecific IgG2 (Figure 30 C) that IgG2_B (Figure 30 B) is formed and IgG2_A and IgG2_B between.

Figure 31 shows IgG2_A, IgG2_B and clicks on the SEC of product.

Specific embodiment

Bispecific antibody (BsAb) is made up of (Fig. 1) two incomplete antibody fragments being connected chemically in hinge area.It is initial anti- Body is IgG1 or IgG4 isotypes.Initial antibodies can contain the hinge area of modification, and wherein Cys residues sport first Ser, Next disulfide bond is only stayed at hinge.The generation of bispecific antibody is related to three key steps.The first step is optionally also Both original antibody A and B are forming incomplete antibody fragment.Second step is by being based on the coupling of cysteine by Functional portions X or Y In being introduced into the hinge area of each incomplete antibody fragment, antibody fragment the halfbody A' and B' of chemical modification is caused respectively.In final step In, two antibody fragment halfbodies are linked together by the chemical bond between X and Y portion, to form bispecific antibody.

Present disclose provides the bispecific for producing chemistry locking from IgG antibody-likes " A " and IgG antibody-likes " B " resists The method of body " AB " or " BA ", it includes：

A () reduction has hinge residues sequence (EU index numbers：Residue 226-229；Kabat is numbered：Residue 239-242) The first antibody " A " of CPPC or CPSC or SPPC or SPSC and with hinge residues sequence (EU index numbers：Residue 226-229； Kabat is numbered：Residue 239-242) CPPC or CPSC or SPPC or SPSC SA " B " it is anti-to form incomplete antibody A and half Body-B, wherein antibody A combine the first target and antibody B combines the second target, and thus reducing condition destruction has hinge residues sequence Two sulphur in any interchain or chain in the hinge area of one antibody-like of row (residue 226-229) CPPC or CPSC or SPPC or SPSC Key；

B () will be connected to one or two Cys residue (EU of the hinge core sequence of incomplete antibody A from the compound of Formulas I Index number：Residue 226 and 229；Kabat is numbered：Residue 239 and 242) to form half-antibody A of connection, it has and is selected from In following structure：

M=N, C

M '=N, C

Z=I, Br, SPh,

Wherein N₃It is-N=N=N；

C () will be connected to one or two Cys residue (EU of the hinge core sequence of antibody B from the compound of Formula II Index number：Residue 226 and 229；Kabat is numbered：Residue 239 and 242) to form the antibody B of connection, its have selected from Under structure：

M=N, C

M '=N, C

Z=I, Br, SPh；With

Preferably, in reducing agent, such as Cys, dithiothreitol (DTT), beta -mercaptoethanol, cysteamine, TCEP (three (2- Carboxyethyl) phosphine), the reduction of antibody A is carried out in 2-MEA (2-MEA) and combinations thereof with formed incomplete antibody A and antibody B also It is former forming incomplete antibody B.Preferably, the hinge area of the antibody A with two Cys residues is selected from the portion of following structure with having Divide A connections：

M=N, C

M '=N, C

Z=I, Br, SPh

Wherein N₃It is-N=N=N.Preferably, the hinge area of the antibody B with two Cys residues is following with being selected from Structure part B connection：

M=N, C

M '=N, C

Z=I, Br, SPh.

The disclosure further provides a kind of bispecific antibody AB of chemistry locking, wherein the incomplete antibody A for connecting

Wherein N₃It is-N=N=N；

It is incorporated into the antibody B of connection

To form the bispecific antibody AB with structure shown in Figure 10.

M=N, C

M '=N, C

Z=I, Br, SPh,

Wherein N₃It is-N=N=N；

With with selected from following structure incomplete antibody B：

M=N, C

M '=N, C

Z=I, Br, SPh.

Preferably, antibody A reduction with formed incomplete antibody A and antibody B reduction with formed incomplete antibody B be in reducing agent such as, L- Cysteine, dithiothreitol (DTT), beta -mercaptoethanol, cysteamine, TCEP (three (2- carboxyethyls) phosphines), 2-MEA (2-MEA) And combinations thereof in carry out.

Preferably, antibody A and B are monoclonal antibodies.Monoclonal antibody can be by hybridoma method or by recombinant DNA Produce with protein expression method.Additionally, antibody A and B are full length antibody or antibody fragment.

Antibody A and B have CPPC core hinges region sequence or CPSC core hinges region sequence or SPPC core hinge region sequences Row or SPSC core hinge region sequence (EU index numbers：Residue 226-229；Kabat is numbered：Residue 239-242).Additionally, step Suddenly (d) incubating the step of also include addition reducing agent, and wherein reducing agent is selected from Cys, dithiothreitol (DTT), β-sulfydryl second Alcohol, cysteamine, TCEP (three (2- carboxyethyls) phosphines), 2-MEA (2-MEA) and combinations thereof.

Conventional biochemical technology such as absorbance measuring, HP-SEC, SDS-PAGE, Native PAGE and RP- can be used HPLC come analyze gained bispecific antibody quality and purity.It should be noted that because the joint of Formulas I is to the joint of Formula II The specificity of compatibility, disclosed method generally avoids any purification step.However, providing in US2010/0105874 Various purification steps, the disclosure of which is incorporated herein by.

The step of disclosed method also includes that preparing bispecific antibody is used for therapeutical uses.This is by the suitable mankind Use, the preparation for being especially suitable for the bispecific antibody of effective dose in the aqueous solution of parenteral or intravenous administration comes real It is existing.

Fig. 2 shows the scheme that bispecific monoclonal antibody (mAb) is produced by chemical coupling.It is as herein described double special Different in nature mAb is made up of two incomplete antibody fragments being connected chemically in hinge area.The process that bispecific mAb is produced includes three masters Want step (Fig. 2).The first step is the selective reduction hinge disulfide bond in two kinds of different mAs b A and B respectively.Second step is to pass through Joint X or Y induce the interior connection of the chain between two cysteines in each mAb on identical heavy chain.Connection procedure is produced in chain MAb fragments A' and B' of two chemistry lockings.In the final step, two mAb fragments are connected by the chemical bond between X and Y It is connected together, to form bispecific antibody AB.

IgG1, wt IgG4 with hinge mutation (CPSC) and the IgG4 with hinge mutation (SPSC) are for this research In.

The first step is to reduce each in antibody A and antibody B.In one embodiment, used in 0.1M PBS pH The 2-MEA (2-MEA) of 10 molar equivalents in 7.4,1.0mM diethylene-triamine pentaacetic acid (DTPA) is at 37 DEG C Process antibody (10mg) 2 hours.Excessive 2-MEA using 50kDa filters centrifuge tube with 3,000RPM centrifugation 20 minutes Fall from the mAb purifying of partial reduction.Three washings altogether are carried out with 0.1M PBS.Protein concentration makes for 1.0mg/mL solution 1.58 absorbance comes quantitative at 280nm, and uses the molecular weight determination molar concentration of 150,000g/mol.

In another embodiment of reduction step, antibody (10mg) is used in into 0.1M PBS pH 7.4,1.0mM bis- The dithiothreitol (DTT) (DTT) of 3.0 molar equivalents in ethylenetriamine pentacetic acid (DTPA) is processed 2 hours at 24 DEG C.It is excessive DTT be centrifuged 20 minutes with 3,000RPM using 50kDa filters centrifuge tube, fall from the mAb of partial reduction purifying.With 0.1M PBS carry out 3 washings altogether.

In another embodiment of reduction step, mAb (10mg) is used in into 0.1M PBS pH 8.0,1.0mM bis- is sub- Three (2- carboxyethyls)-phosphine (TCEP) of 2.0 molar equivalents in ethyl pentaacetic acid (DTPA) are processed 2 hours at 24 DEG C. MAb concentration is 8.0mM.In the case where not purifying, the mAb of partial reduction is directly used in coupling step.

Second step is coupling step.The mAb " antibody A " of the partial reduction from reduction step in 0.1M PBS is added Enter in the crosslinking agent Z-X-Z of 2.5 molar equivalents (Fig. 2 and Fig. 3).Crosslinking agent takes from pre-prepared DMSO stock solution (1mg/ mL).In the reactive mixture, partial reduction AC is 8.0mg/mL, and DMSO contents are 5% (v/v).Enter at 24 DEG C Row is coupled 2 hours.Any unreacted excess cross-linker is quenched with cysteine (1mM final concentrations).Using phosphate-buffered The mAb that the PD-10 posts purifying of salt solution balance is coupled.The mAb structures of coupling are shown in Figure 4.Under the same conditions, by the 2nd mAb (antibody B) is coupled and purifies with crosslinking agent Z-Y-Z (Fig. 5 and Fig. 6).The mAb structures of coupling are shown in Fig. 7 and Fig. 8.

3rd step is interchain coupling step.It has been illustrated in Figure 9 the click coupling for interchain linkage.In short, for The antibody fragment (3.0mg) of the Azide modification in 0.5mL PBS (0.1M, pH 7.4), adds 0.5mL PBS (0.1M, pH 7.4) antibody fragment of the alkynes modification of the 3.0mg in.The final content that 50 μ L acetonitriles and acetonitrile are added in the mixture is 5% (v/v).After reacting 3 hours at room temperature, it is centrifuged 20 minutes under 3,000RPM to purify using 100kDa filter centrifuge tubes Mixture.Mixture is washed 3 times with PBS, and products therefrom is carried out into vitro characterization.

Embodiment 1

The embodiment indicates the synthesis of the bispecific antibody according to disclosed method.Fig. 4 show by with IgG The chemical coupling of two Cys residues in the hinge area of antibody-like produces the scheme of bispecific monoclonal antibody (mAb).Institute is public The bispecific mAb for opening is made up of two incomplete antibody fragments being connected chemically at its each hinge area.Synthesis bispecific mAb Method include Fig. 5 shown in three key steps.The first step be respectively in two kinds of different mAb (A and B) optionally Reduction hinge disulfide bond.Second step be by joint X or Y induce in each mAb two cysteines on identical heavy chain it Between chain in connection.Connection procedure produces mAb fragments A' and B' of two chemistry lockings in chain.In the final step, two mAb Fragment is joined together to form bispecific antibody AB by the chemical bond between X and Y.

More specifically, we obtain antibody " A ", one kind has hinge mutation (CPSC IgG1), and antibody " B ", one Plant wild type IgG4.The first step is antibody reduction.Condition 1：Antibody (10mg) is individually used in 0.1M PBS pH 7.4, It is little that the 2-MEA (2-MEA) of 10 molar equivalents in 1.0mM diethylene-triamine pentaacetic acids (DTPA) processes 2 at 37 DEG C When.Excessive 2-MEA is using 50kDa filters centrifuge tube with 20 minutes being centrifuged under 3,000RPM from the mAb of partial reduction Purifying falls.Three washings altogether are carried out with 0.1M PBS.For the absorbance pair that 1.0mg/mL solution uses at 280nm 1.58 Protein concentration is carried out quantitatively, and uses the molecular weight determination molar concentration of 150,000g/mol.

Condition 2：3.0 moles in 0.1M PBS pH 7.4,1.0mM diethylene-triamine pentaacetic acids (DTPA) are worked as The dithiothreitol (DTT) (DTT) of amount processes antibody (10mg) 2 hours at 24 DEG C.Excessive DTT uses 50kDa filter centrifuge tubes Fall from the mAb purifying of partial reduction with being centrifuged 20 minutes under 3,000RPM.3 washings altogether are carried out with 0.1M PBS.

Condition 3：2.0M mole in 0.1M PBS pH 8.0,1.0mM diethylene-triamine pentaacetic acids (DTPA) is worked as Three (2- carboxyethyls)-phosphine (TCEP) of amount process mAb (10mg) 2 hours at 24 DEG C.MAb concentration is 8.0mM.What is do not purified In the case of, the mAb of partial reduction is directly used in coupling.

Embodiment 2

The embodiment shows that the bispecific antibody for preparing in embodiment 1 remains two kinds of combinations of its original half Mab Feature.

The synthesis of 1- (2- (2- nitrine base oxethyls) ethyl) the bromo- 1H- pyrrole-2,5-diones of -3,4- two：

It is added dropwise to the bromo- 1H- pyrrole-2,5-diones (10mmol) of 2.5g 3,4- bis- in 60mL THF and 1g NMM MeOCOCl (10mmol, 940mg, in 10ml DCM), stirs 20 minutes, then dilutes reaction solution with 60mL DCM, uses Water washing 3 times, organic phase is stirred with anhydrous sodium sulfate, and concentration obtains 2.65g 3, bromo- 2, the 5- dioxos -2H- pyrroles of 4- bis- Cough up -1 (5H)-carboxylic acid methyl ester.To 311mg, add in the 1mmol compounds 2- (2- nitrine base oxethyls) ethamine (130mg, 1mmol) show to react and completed in 20 minutes with 5mL DCM, TLC, then extracted by DCM and salt solution, use NH₄Cl solution is washed Wash, with anhydrous sodium sulfate drying, and and then concentrate for post purifying, use 2:1 hexane and ethyl acetate flash distillation, obtain 230mg's 1- (2- (2- nitrine base oxethyls) ethyl) the bromo- 1H- pyrrole-2,5-diones of -3,4- two.¹HNMR：3.32ppm (t, J= 5.0Hz, 1H), 3.40ppm (t, J=5.0Hz, 1H), 3.50ppm (q, J=5.0Hz, 1H), 3.62ppm (t, J=, 1H), 3.63-3.69ppm (m, 3H), 3.84ppm (t, J=5Hz, 1H).Fw：365.9, C₈H₈Br₂N₄O₃；Mass spectra peak (1:2:1)： 366.9,368.9,370.9。

Embodiment 3

This embodiment illustrates and carry out the double spies of chemical generation using the single Cys residues in the hinge area of IgG antibody-likes Heterogenetic antibody.Initial mAb as herein described contains the hinge area of engineering, wherein a Cys on each bar chain at same position Ser is sported, so as to produce the hinge for only leaving single disulfide bond.The process that bispecific mAb is produced includes three main steps Suddenly (Fig. 1).The first step is optionally to reduce hinge disulfide bond in two kinds of different mAb A and B respectively.Second step is to pass through Coupling based on cysteine introduces functional groups X or Y.Cys- Connection Steps produce mAb fragments A' of two chemistry lockings And B'.In the final step, two mAb fragments are joined together to form bispecific and resist by the chemical bond between X and Y Body AB.In our current research using have hinge region mutations (SPPC IgG1 monoclonal antibodies).

Condition 1：10 molar equivalents in 0.1M PBS pH 7.4,1.0mM diethylene-triamine pentaacetic acids (DTPA) 2-MEA (2-MEA) process antibody (10mg) 2 hours at 37 DEG C.Excessive 2-MEA is centrifuged using 50kDa filters Pipe falls with being centrifuged 20 minutes under 3,000RPM from the mAb purifying of partial reduction.Three washings altogether are carried out with 0.1M PBS.It is right Carry out quantifying protein concentration using at 280nm 1.58 absorbance in 1.0mg/mL solution, and use 150,000g/ The molecular weight determination molar concentration of mol.

Condition 2：3.0 moles in 0.1M PBS pH 7.4,1.0mM diethylene-triamine pentaacetic acids (DTPA) are worked as The dithiothreitol (DTT) (DTT) of amount processes antibody (10mg) 2 hours at 24 DEG C.Excessive DTT uses 50kDa filter centrifuge tubes Fall from the mAb purifying of partial reduction with being centrifuged 20 minutes under 3,000RPM.3 washings altogether are carried out with the PBS of 0.1M.

Embodiment 4

This example shows and bispecific antibody is prepared and with by each incomplete antibody piece according to method disclosed herein The disclosed method for being connected chemically structure that the hinge area of section is connected to each other.Antibody scaffold is：

With hinge mutation (SPSC IgG4).

We change each Ig antibody to produce incomplete antibody by chemical modification first.Specifically, buffer-exchanged reaction will Antibody (0.5-3mg) is added in the filter centrifuge tube (Millipore, UFC903024) of 15mL, and adds the pH of appropriate amount 8.0 PBS 1mM DTPA (diethylene-triamine pentaacetic acid) buffer solutions to the 50mL on pipe is marked.By pipe at 5 DEG C with 3, 000RPM is centrifuged 20 minutes.Antibody is transferred in 1.5mL plastic jars and is used Nanodrop (Fisher, ND-2000UV- Vis spectrophotometers) check concentration.Final AC is between 5-8mg/mL.

Prepare 1mg/mL TCEP ((three (2- carboxyethyls) phosphines)) in pH 8.0PBS (1.0mM DTPA) buffer solution The stock solution of (Sigma-Aldrich, C4706).The TCEP solution that we need to be added in antibody using following table calculating Volume, this depends on the equivalents and final mass of the antibody reclaimed after buffer-exchanged.

During the TCEP solution (being calculated by upper table) of appropriate amount is added into antibody-solutions, gently it is vortexed, and bottle is put On rotating disk.Reduction reaction is carried out at room temperature 90 minutes.

Based on upper table calculating prepare DMSO (Sigma-Aldrich, 472301) in DBCO- maleimides (Click Chemistry Tools, A108-100) stock solution.DBCO- maleimides in DMSO are added into antibody In sample (purifying without TCEP).The final quantity of DMSO is for about 5% (v/v) in antibody samples.At room temperature by rotating disk Coupling reaction is carried out into 1 hour under mixing condition.By each sample be placed in single 15mL filters centrifuge tube (Millipore, UFC903024 in), and 1X DPBS (Corning, 21-031-CM, without the calcium or magnesium) buffer solution of appropriate amount is added to pipe 50mL is marked.Sample is centrifuged 20 minutes with 3,000RPM at 5 DEG C.Repeated washing step again.After washing, sample is shifted To in single 1.5mL plastic jars, in being placed in refrigerator (5 DEG C).

For IgG4 antibody, the TCEP of 4.0 equivalents provides substantial amounts of incomplete antibody.For IgG1 antibody, 3.5 equivalents TCEP provides substantial amounts of incomplete antibody.For two kinds of antibody uses the DBCO of 5.0 equivalents.

DMSO is added in in DBCO- maleimides (1.0mg, the 1.0 equivalents) solution in DMSO (0.12mL) (0.6mL) azido-PEG4- azide (2.5mg, 5.0 equivalents) in.Mixture is stirred at room temperature into 2 hours.As led to Cross reaction shown in LC/MS to complete.The molecular weight of gained azide-maleimide is 627.65g/mol.Azide-horse Carry out acid imide synthesis (scheme 1)：

The synthesis of 1. azide of scheme-maleimide

During azide-maleimide (5.0 equivalent) in DMSO is added into antibody samples.DMSO in antibody samples Final quantity be for about 5% (v/v).Coupling reaction is carried out at room temperature 1 hour under by the mixing of rotating disk.Wash as previously mentioned Wash sample.

Half and half antibody fragment is purified by hydrophobic interaction post (HIC).HIC analyses are existed using TOSOH butyl-NPR posts Carry out under 40 DEG C of column temperatures and 0.6mL/min flow velocitys.The salinity reduced in 50mM sodium phosphate buffers (pH 7.0) is (from 1.5 To 0M ammonium sulfate) and increase organic modifiers (from 0% to 25% isopropanol) 30 minutes gradients realize wash-out.By SDS PAGE analyzes incomplete antibody fragment.Specifically, for each sample to be analyzed, 20 μ L of 0.6mg/mL concentration are needed.We abide by Follow and set up scheme operation PAGE gel (RTP AD001-01 and AD002-01).Fig. 9 shows the incomplete antibody piece of chemical modification The non-reduced SDS PAGE of section (swimming lane 2 and 3).

Embodiment 5

This embodiment illustrates and bispecific antibody is produced by click-reaction.Figure 10 is shown by two incomplete antibody fragments Between click be coupled produce bispecific antibody.Click-reaction between two incomplete antibody fragments is shown in Figure 10.To in PBS In incomplete antibody azide fragment (500 μ g) (5.0mg/mL) in add PBS in incomplete antibody-DBCO fragments (500 μ g) (5.0mg/mL).Reaction carries out 2 hours under by the mixing condition of rotating disk at room temperature.Mixture is carried out into SDS PAGE point Analyse (Figure 11) and by ion-exchange chromatogram purification.

It is anti-with 0.6mL/min flow velocitys purifying bispecific using Thermo WCX-10 posts on Agilent 1200HPLC Body.30 minutes gradients of the salinity (from 0 to 100mM NaCl) increased in the 10mM MES buffer solutions of pH 5.7 are realized Wash-out.By IdeS protease digestion bispecific antibody STI CBA-0710, and on Water Xevo G-2QTOF mass spectrums It is analyzed and confirms (Figure 12).

The biology of bispecific antibody STI CBA-0710 is determined by the size exclusion chromatography (SEC) of bispecific antibody Physical property (Figure 13).Specifically, on Agilent 1200HPLC using tsk gel SuperSW3000 posts (4.6mm ID × 30cm, 4 μm) analysis bispecific antibody.Buffer solution be 0.2M potassium phosphates, 0.25M KCl, pH 6.2.

The SEC data of the bispecific antibody CBA-0710 of table 2.

It is main	HMWS	LMWS
			98.3%	1.2%	0.5%

Figure 14 shows combinations of the bispecific antibody CBA-0710 on BIAcore.Using standard NHS/EDC coupling method First antigen is fixed on into CM5 sensor chips up to about 1500RU.Running buffer is used as baseline.Loading bispecific resists Body, then buffer solution, then carries out the combination for the second antigen.

Embodiment 6

This example shows the various measurement results of the bispecific antibody for producing herein.There is bispecific antibody Combination and function based on cell.Bispecific antibody CBA-0710 combines MDA-MB-231 (human breast carcinoma) cell (Figure 15), As by Flow Cytometry Assay.Determine the EC of antibody₅₀Value.With reaching without enzyme cell dissociation buffer solution (GIBCO) yield table Antigen -1 and antigen -2 both MDA-MB-231 triple negative breast cancers (TNBC) cells, and be transferred to the orifice plate of v-shaped bottom 96 (50, 000 cells/well).By cell and FACS buffer solution (PBS+2%FBS)+NaN₃In bispecific antibody CBA-0710 or Parent's monospecific antigen -1 or the antibody of antigen -2 are serially diluted thing in incubated on ice 45 minutes.In FACS buffer solution After washing 2 times, 1 is added：The phycoerythrin of 1000 dilutions is coupled anti-human igg (γ-chain specificity), and incubates 30 minutes. After finally washing, on Intellicyt high flux flow cytometers (HTFC) fluorescence intensity is measured.Using Graphpad Prism softwares and nonlinear regression and fitting analyze data.Data point is shown as the median fluorescence intensity (MFI) of positive-labeled cells +/- standard error.EC₅₀Value is reported as reaching the AC with 50% maximum combined of cell.

As a result in being shown in table 3 and Figure 15, and show that bispecific antibody is thin with MDA-MB-231 compared with parental form The combination of born of the same parents improves, with the sub- nanomole EC similar to the antibody of antigen -2₅₀Value, and as the antibody of antigen -1 it is high Bond strength.

The combination data of the bispecific antibody of table 3. and parental type antibody

Show the antagonistic activity of bispecific antibody CBA-0710.Specifically, according toPhospho- Met (panTyr) Sandwich ELISA kits #7333 scheme runs bispecific antibody CBA-0710 to c-MET phosphorylations Suppression.In short, cell lysate being added into the detection antibody of reconstruct and being incubated, two grades of the HRP- joints of reconstruct are subsequently adding Antibody.After washing, add tmb substrate and incubate.After adding STOP solution, result is read.Figure 16 shows bispecific antibody Antagonistic activities of the CBA-0710 in triple negative breast cancer cell.STI-A0607 is the monoclonal antibody of antigen -1, and STI- A1010 is the monoclonal antibody of antigen 2.HGF is the native ligand of antigen -1.

There is the immunoregulatory activity of bispecific antibody CBA-0710.Adjust to measure bispecific antibody CBA-0710 The ability of section t cell responses, the CD4+ cells of purifying are cultivated together with allogene dendritic cells, and it is by GM- Monocyte 7 days is cultivated in CSF and IL-4 and prepare.Arrange parallel flat with allow the 3rd day and the 5th day collection supernatant and IL-2 and IFN γ are measured respectively using commercial ELISA Kit.In humanization antigen -2 (immunologic test point) mAb of competition Portion produces and as positive control IgG1, and incoherent STI people mAb is used as negative control IgG antibody.Figure 17 shows response Increase in the IFN-γ release of bispecific antibody CBA-0710.STI-A1010 is antigen -2 (immunologic test point) monoclonal Antibody.Competition mAb is humanization antigen -2 (immunologic test point) monoclonal antibody.

Embodiment 7

This embodiment illustrates using F (ab) '₂Antibody A ' and the disclosed bispecific antibody of B' synthesis scheme.This Bispecific F (ab) ' described in text₂It is made up of (Figure 20) two F (ab) ' fragments being connected chemically in hinge area.Starting antibody It is IgG1 or IgG4 isotypes.Starting antibody contains the hinge area of modification, and wherein Cys residue mutations are Ser residues, so as in hinge Sequence only stays next disulfide bond.Bispecific F (ab) '₂The generation of chemistry locking bispecific antibody is related to four main steps Suddenly.The first step is to remove Fc fragments.Second step is difference selective reduction antibody A and B.3rd step is by based on cysteine Be coupled in hinge area introducing Functional portions X or Y, so as to produce the antibody fragment A' and B' of chemical modification respectively.Most In latter step, two antibody fragments are joined together to form bispecific F (ab) ' by the chemical bond between X and Y₂.Should The scheme of synthesis is shown in Figure 21.

Specifically, We conducted with have hinge mutation (SPPC IgG1A antibody) and with hinge mutation (SPSC) IgG4 B antibody synthesis F (ab) '₂The method of chemistry locking bispecific antibody.Using enzyme IdeS (its be only hinge area it Under a specific position cut IgG digestive ferment), antibody (1.5mg) is added into each of IdeS (A0-FR1-008) Guan Zhong, and be incubated overnight on convertible shaking table (head to head spinner) at 37 DEG C.Then Protein A purification is used Remove Fc fragments.

Antibody (1-10mg) is added in 15mL filter centrifuge tubes (Millipore, UFC903024), and is added suitable The 50mM sodium phosphates of equivalent, 150mM NaCl, 5mM EDTA, the 50mL marks on the buffer solutions of pH 7.7 to pipe.By pipe at 22 DEG C Under with 3,000RPM be centrifuged 20 minutes.Antibody is transferred in 1.5mL plastic jars, and using Nanodrop (Fisher, ND- 2000UV-Vis spectrophotometers) check concentration.Final AC is up to 10mg/mL.

The 50mM sodium phosphates of addition 1mL in a bottle containing 6mg 2-MEA HCl, 150mM NaCl, The buffer solution of 5mM EDTA, pH 7.7 (obtains 50mM 2-MEA).Add 50mM 2-MEA to F (ab) '₂Final concentration 15mM, fully Mixing.Incubate 15 minutes at 37 DEG C.Using NAP-5 (GE17-0853-02) desalting columns from reduction F (ab) '₂Separate 2- MEA。

Prepare the 1mg/mL TCEP ((three (2- carboxyethyls) phosphines)) in PBS (1.0mM DTPA) buffer solution of pH 8.0 The stock solution of (Sigma-Aldrich, C4706).Depending on the F (ab) ' reclaimed after Protein A purification₂Equivalents and final Quality, the TCEP of 5 equivalents is added in the F of desalination (ab) ', shake well, and is incubated 5 minutes at room temperature.

For DBCO (dibenzo cyclooctyl)-maleimides and azide-maleimide amine coupling, DBCO- is prepared Maleimide (Click Chemistry Tools, A108-100) DMSO (Sigma-Aldrich, 472301) in storage Standby solution, and the DBCO- maleimides of 20 equivalents in DMSO are added into F (ab) ', and (A) sample (does not have TCEP's Purifying) in.The final quantity of DMSO is for about 5% (v/v) in antibody samples.At room temperature coupling reaction is carried out by the mixing of rotating disk 2 hours.Azide-maleimide (20 equivalent) in DMSO is added into F (ab) ' (B) in sample.In antibody samples The final quantity of DMSO is for about 5% (v/v).At room temperature coupling reaction is carried out 2 hours by the mixing of rotating disk.

For washing step, by each sample be placed in single 15mL filters centrifuge tube (Millipore, UFC903024 in), and 1X DPBS (Corning, 21-031-CM, without the calcium or magnesium) buffer solution of appropriate amount is added to pipe 50mL is marked.Sample is centrifuged 20 minutes at 22 DEG C with 3,000RPM.Repeated washing step again.After washing, sample is turned In moving on to single 1.5mL plastic jars, it is placed in refrigerator (5 DEG C) or for clicking on step.For F (ab) ' fragment analysis, Using SDS PAGE programs.For each sample to be analyzed, the 20 μ L that concentration is 0.6mg/mL are needed.Follow the side for having set up Case operation PAGE gel (RTP AD001-01 and AD002-01) is (Figure 21).

Click-reaction scheme between two F (ab) ' fragments is shown in Figure 23.To F (ab) '-azide piece in PBS Section (500 μ g) (5.0mg/mL) in add PBS in F (ab) '-DBCO fragments (500 μ g) (5.0mg/mL).Pass through at room temperature The mixing of rotating disk is reacted overnight.Mixture is carried out into SEC analyses (Figure 24).

The biophysics of the bispecific antibody prepared in the present embodiment using size exclusion chromatography (SEC) analysis Matter.Using size exclusion chromatography (SEC) Agilent of tsk gel SuperSW3000 posts (4.6mm ID × 30cm, 4 μm) 1200HPLC analyzes F (ab) ' _ A, F (ab) ' _ B and bispecific click _ F (ab) '₂(Figure 24).Buffer solution 0.2M potassium phosphates, 0.25M KCl, pH 6.2.

Bispecific F (ab) ' is confirmed by mass spectrography₂.Bispecific F is analyzed on Water Xevo G-2 QTOF9 (ab)'₂(Figure 25).Using size exclusion chromatography (SEC) purifying bispecific F (ab) '₂.Using tsk gel SuperSW3000 posts Size exclusion chromatography (SEC) the Agilent1200 HPLC of (4.6mm ID × 30cm, 4 μm) are used for purifying bispecific click _ F (ab)'₂(Figure 26).Buffer solution 0.2M potassium phosphates, 0.25M KCl, pH 6.2.

External affinity measurement is the measurement (Figure 27) for using Octet Red.Sensors A R2G is used to measure Octet Red Bispecific F (ab) ' on (ForteBio, Inc.)₂AI.In short, measurement scheme is as follows：300 seconds baselines； 300 seconds 10 μ g/ml bispecific F (ab) ' of loading₂, 120 seconds baselines；300 seconds antigen As；Dissociate within 300 seconds；300 seconds antigen B and Dissociation (Figure 27) in 300 seconds.Enter line sensor hydration and baseline-and dissociation measurement in PBS.

Embodiment 8

This embodiment illustrates using IgG2 antibody As ' and the disclosed bispecific antibody of B' synthesis scheme.This paper institutes The bispecific IgG2 for stating is made up of (Figure 29) two IgG2 fragments being connected chemically in hinge area.Starting antibody is IgG2 of the same race Type.The generation of bispecific IgG2 is related to three key steps.The first step be reduce in IgG2 antibody hinge regions (in four ) one or two disulfide bond, it remains in that homodimer structure.Second step is by being coupled at based on cysteine Functional portions X or Y are introduced in hinge, so as to cause the antibody fragment A' and B' of chemical modification respectively.In the final step, lead to Two kinds of antibody are joined together to form bispecific IgG2 by the chemical bond crossed between X and Y.

Specifically, We conducted the method for synthesizing the bispecific antibody of IgG2 chemistry lockings.By antibody (1- In 10mg) being added to 15mL filter centrifuge tubes (Millipore, UFC903024), and add appropriate amount 50mM sodium phosphates, 150mM NaCl, 5mM EDTA, the 50mL marks on the buffer solutions of pH 7.7 to pipe.Pipe is centrifuged into 20 at 22 DEG C with 3,000RPM Minute.Antibody is transferred in 1.5mL plastic jars, and using Nanodrop (Fisher, ND-2000UV-Vis spectrophotometrics Meter) check concentration.Final AC is up to 10mg/mL.

Prepare TCEP ((three (the 2- carboxy ethyls) of the 1mg/mL in PBS (2.0mM DTPA) buffer solution of pH 8.0 Phosphine)) stock solution of (Sigma-Aldrich, C4706).According to the equivalents and gained quality of IgG2, by the TCEP of 2 equivalents In being added to IgG2 solution, fully shake and at room temperature incubation 90 minutes.

DBCO (dibenzo cyclooctyl)-maleimides and azide-maleimide

In order to be coupled, DBCO- maleimides (Click Chemistry Tools, A108-100) are prepared in DMSO (Sigma-Aldrich, 472301) in stock solution, and the DBCO- maleimides of 5 equivalents in DMSO are added to IgG2_A samples (purifying without TCEP).The final quantity of the DMSO in antibody samples is for about 5% (v/v).Pass through at room temperature The mixing of rotating disk carries out 1 hour coupling reaction.Azide-maleimide (5 equivalent) in DMSO is added into IgG2 (B) In sample.The final quantity of DMSO is for about 5% (v/v) in antibody samples.Carry out 1 hour at room temperature being coupled by the mixing of rotating disk Reaction.

For washing step, by each sample be placed in single 15mL filters centrifuge tube (Millipore, UFC903024 in), and 1X DPBS (Corning, 21-031-CM, without the calcium or magnesium) buffer solution of appropriate amount is added to pipe 50mL is marked.Sample is centrifuged 20 minutes at 22 DEG C with 3,000RPM.Repeated washing step again.After washing, sample is turned In moving on to single 1.5mL plastic jars, it is placed in refrigerator (5 DEG C) or for clicking on step.

For each sample to be analyzed, 20 μ L of 0.6mg/mL concentration are needed.Follow the scheme operation SDS- for having set up PAGE gels (RTP AD001-01 and AD002-01) are (Figure 29).

For mass spectrum, antibody A is reduced with TCEP, and be coupled with DBCO, be analyzed on Water Xevo G-2QTOF. 2 DBCO of as shown by data (only one disulfide bond reduction) or 4 DBCO (two disulfide bond reductions) are coupled with our IgG2 (Figure 30 A-C).

The biophysics of the bispecific antibody prepared in this embodiment using size exclusion chromatography (SEC) analysis Matter.Using the size exclusion chromatography (SEC) of tsk gel SuperSW3000 posts (4.6mm ID × 30cm, 4 μm) Agilent1200HPLC is analyzing bispecific IgG2 (Figure 31).Buffer solution 0.2M potassium phosphates, 0.25M KCl, pH 6.2.

Claims

1. one kind is used for the method for preparing bispecific antibody " AB " or " BA " from first antibody " A " and SA " B ", its bag Include：

A () makes the first antibody A and reducing agent in the essentially all disulfide bond that be enough to cut in hinge area between heavy chain Under the conditions of contact to produce a pair of first antibody fragments A', each first antibody fragment A' is comprising being connected to the single of single heavy chain Light chain, wherein the heavy chain has the reactive sulfydryl that one or more are formed by the reduction of the disulfide bond；

B first Heterobifunctional joint is connected to first antibody fragment A' by (), the first Heterobifunctional joint includes (i) For the first sulfydryl reactive functional groups for being covalently attached with the reactive sulfydryl of the heavy chain of the first antibody fragment and (ii) azide, so as to form the first antibody fragment of azide functionalization；

C () makes the SA B and reducing agent in the essentially all disulfide bond that be enough to cut in hinge area between heavy chain Under the conditions of contact to produce a pair of SA fragments B', each SA fragment B' is comprising being connected to the single of single heavy chain Light chain, wherein the heavy chain has the reactive sulfydryl that one or more are formed by the reduction of the disulfide bond；

D second Heterobifunctional joint is connected to SA fragment B' by (), the second Heterobifunctional joint includes (i) For the second sulfydryl reactive functional groups for being covalently attached with the reactive sulfydryl of the heavy chain of the SA fragment and (ii) alkynes；So as to form the SA fragment of alkynes functionalization；With

E () makes the first antibody fragment of the azide functionalization react with the SA fragment of the alkynes functionalization, with The first antibody fragment is covalently attached to by the SA fragment by cycloaddition of the azide to the alkynes, So as to form the bispecific antibody " AB " or " BA " of chemistry locking.

2. it is according to claim 1 for from first antibody " A " and SA " B " prepare bispecific antibody " AB " or The method of " BA ", wherein the first Heterobifunctional joint has Q-L-N₃Form, wherein Q be comprising alkyl halide, benzyl halide, The sulfydryl reactive functional groups of maleimide, halo maleic amide or dihalo maleimide；And L is former with 3-60 The hydrocarbon joint of son, and N₃It is azido.

3. it is according to claim 1 for from first antibody " A " and SA " B " prepare bispecific antibody " AB " or The method of " BA ", wherein the first Heterobifunctional joint has Q-L-N₃Form, wherein Q is comprising maleimide, halogen For maleic amide or the sulfydryl reactive functional groups of dihalo maleimide base group；It is with unit-(CH with L₂CH₂-O )_n- and/or-(O-CH₂CH₂)_n- polymer configuration in there is the hydrocarbon joint of 3-60 atom, wherein " n " is independently 1-20 Integer；And N₃It is azido.

4. it is according to claim 2 for from first antibody " A " and SA " B " prepare bispecific antibody " AB " or The method of " BA ", wherein first heterobifunctional linker has following form：

Wherein

Q is the sulfydryl reactive group of following form：

Independently selected from H, Br, I and SPh, it is not H that condition is at least one Z to wherein Z；It is independently CR* or N with M；

Wherein X₁、X₂、X₃、X₂、X₄And X₅Independently selected from key ,-O- ,-NR^N- ,-N=C- ,-C=N- ,-N=N- ,-CR*=CR*- (cis or trans) ,-C ≡ C- ,-(C=O)-,-(C=O)-O- ,-(C=O)-NR^N- ,-(C=O)-(CH₂)_n- ,-(C=O)- O-(CH₂)_n- ,-(C=O)-NR^N-(CH₂)_n- and-(C=O)-NR^N-(CH₂CH₂-O)_n-, wherein " n " is 0 or the integer of 1-10；

Wherein R^a、R^b、R^cAnd R^dIndependently selected from-O-,-NR^N–、–CH₂–、–(CH₂)_n–、–(CR*₂)_n–、–(CH₂CH₂–O)_n–、– (CR*₂CR*₂–O)_n–、–(O–CH₂CH₂)_n–、–(O–CR*₂CR*₂)_n- ,-CR*=CR*-(cis or trans) ,-N=C-,-C= N-,-N=N-,-C ≡ C-,-(C=O)-,-(CH₂)_n- (C=O)-,-(C=O)-(CH₂)_n–、–(CH₂)_n- (C=O)- (CH₂)_n- ,-O-(C=O)-,-(C=O)-O-,-O-(C=O)-O-,-(CH₂)_n- (C=O)-O-,-O-(C=O)-(CH₂)_n、– (C=O)-O-(CH₂)_n–、–(CH₂)_n- O-(C=O)-,-(CH₂)_n- (C=O)-O-(CH₂)_n–、–(CH₂)_n- O-(C=O)- (CH₂)_n–、–NR^N- (C=O)-,-(C=O)-NR^N–、–NR^N- (C=O)-O-,-O-(C=O)-NR^N–、–NR^N- (C=O)- NR^N–、–(CH₂)_n- (C=O)-NR^N–、–NR^N- (C=O)-(CH₂)_n,-(C=O)-NR^N–(CH₂)_n–、–(CH₂)_n–NR^N- (C= O)–、–(CH₂)_n- (C=O)-NR^N––(CH₂)_n–、–(CH₂)_n–NR^N- (C=O)-(CH₂)_n- ,-(C=O)-NR^N–(CH₂CH₂–O )_n–、–(CH₂CH₂–O)_n- (C=O)-NR^N–、–(CH₂)_n- (C=O)-NR^N–(CH₂CH₂–O)_n–、–(CH₂CH₂–O)_n- (C=O)- NR^N–(CH₂)_n- or 2-8 units cyclic hydrocarbon, heterocycle, aryl or heteroaryl ring；Wherein " n " independently is 0 or 1-10 integer； Wherein " l ", " p ", " q " and " r " independently is 0 or 1-10 integer；

Ω is key or C_3-26Hydrocarbon ring or carbocyclic fused ring system, optionally comprising at most four fused rings, wherein each ring has 3-8 Unit and optionally in each ring comprising 1-4 selected from O, S and N hetero atom；

Wherein R^*And R^NIt independently is H or C_1-12Hydrocarbon, is optionally replaced by the 1-6 hetero atom selected from halogen, O, S and N；With

Wherein R^*And/or R^N3-8 yuan of rings can together be formed.

5. it is according to claim 4 for from first antibody " A " and SA " B " prepare bispecific antibody " AB " or The method of " BA ", wherein Q are maleimide, bromo-maleimide or two bromo maleimides.

6. it is according to claim 1 for from first antibody " A " and SA " B " prepare bispecific antibody " AB " or The method of " BA ", wherein the second Heterobifunctional joint has a form of Q-L-G, wherein Q be comprising alkyl halide, benzyl halide, The sulfydryl reactive functional groups of maleimide, halo maleic amide or dihalo maleimide；And L is former with 3-60 The hydrocarbon joint of son, and G is containing ethynylene group.

7. it is according to claim 1 for from first antibody " A " and SA " B " prepare bispecific antibody " AB " or The method of " BA ", wherein G are-C ≡ CH.

8. it is according to claim 1 for from first antibody " A " and SA " B " prepare bispecific antibody " AB " or The method of " BA ", wherein G include the C8 rings with-C ≡ C- keys.

9. it is according to claim 8 for from first antibody " A " and SA " B " prepare bispecific antibody " AB " or The method of " BA ", wherein G have following form：

10. according to claim 8 for preparing bispecific antibody " AB " from first antibody " A " and SA " B " Or the method for " BA ", wherein G has following form：

11. is according to claim 1 for preparing bispecific antibody " AB " from first antibody " A " and SA " B " Or the method for " BA ", wherein the second Heterobifunctional joint has the form of Q-L-G, wherein Q is comprising maleimide, halogen For maleic amide or the sulfydryl reactive functional groups of dihalo maleimide base group；L is with 3-60 atom and comprising tool There is unit-(CH₂CH₂–O)_n- or-(O-CH₂CH₂)_n- polymer hydrocarbon joint, wherein " n " is independently the integer of 1-20；And G is C_8-20Hydrocarbon, it includes the C8 rings with the-C ≡ C- keys that 1,3 Dipolar Cycloadditions can occur with azide.

12. according to claim 10 for preparing bispecific antibody from first antibody " A " and SA " B " The method of " AB " or " BA ", wherein second heterobifunctional linker has following form：

Wherein,

Q is the sulfhydryl reactive group of following form：

G is C_8-20Hydrocarbyl group, it is included with-C ≡ the C- that 1,3 Dipolar Cycloadditions can occur with described azide The C8 rings of key；

X₁、X₂、X₃、X₂、X₄And X₅When occurring every time independently selected from key ,-O-,-NR^N- ,-N=C-,-C=N-,-N=N-,- CR*=CR*-(cis or trans) ,-C ≡ C-,-(C=O)-,-(C=O)-O-,-(C=O)-NR^N–、–NR^N- (C=O)-,- NR^N- (C=O)-O-,-(C=O)-(CH₂)_n- ,-(C=O)-O-(CH₂)_n- ,-(C=O)-NR^N–(CH₂)_n- and-(C=O)- NR^N–(CH₂CH₂–O)_n-, wherein " n " is 0 or the integer of 1-10；

R^a、R^b、R^cAnd R^dIndependently selected from-O-,-NR^N–、–CH₂–、–(CH₂)_n–、–(CR*₂)_n–、–(CH₂CH₂–O)_n–、–(CR*₂CR*₂–O)_n–、–(O–CH₂CH₂)_n–、–(O–CR*₂CR*₂)_n- ,-CR*=CR*-(cis or trans) ,-N=C-,-C=N-,-N =N-,-C ≡ C-,-(C=O)-,-(CH₂)_n- (C=O)-,-(C=O)-(CH₂)_n–、–(CH₂)_n- (C=O)-(CH₂)_n–、–O– (C=O)-,-(C=O)-O-,-O-(C=O)-O-,-(CH₂)_n- (C=O)-O-,-O-(C=O)-(CH₂)_n,-(C=O)-O- (CH₂)_n–、–(CH₂)_n- O-(C=O)-,-(CH₂)_n- (C=O)-O-(CH₂)_n–、–(CH₂)_n- O-(C=O)-(CH₂)_n–、–NR^N– (C=O)-,-(C=O)-NR^N–、–NR^N- (C=O)-O-,-O-(C=O)-NR^N–、–NR^N- (C=O)-NR^N–、–(CH₂)_n–(C =O)-NR^N–、–NR^N- (C=O)-(CH₂)_n,-(C=O)-NR^N–(CH₂)_n–、–(CH₂)_n–NR^N- (C=O)-,-(CH₂)_n- (C= O)–NR^N––(CH₂)_n–、–(CH₂)_n–NR^N- (C=O)-(CH₂)_n- ,-(C=O)-NR^N–(CH₂CH₂–O)_n–、–(CH₂CH₂–O)_n– (C=O)-NR^N–、–(CH₂)_n- (C=O)-NR^N–(CH₂CH₂–O)_n–、–(CH₂CH₂–O)_n- (C=O)-NR^N–(CH₂)_n- or 2- 8 yuan of cyclic hydrocarbons, heterocycle, aryl or heteroaryl rings；Wherein " n " independently is 0 or 1-10 integer；Wherein " l ", " p ", " q " " r " independently is 0 or 1-10 integer；

Ω is independently key or for C_3-26Hydrocarbon ring or carbocyclic fused ring system, optionally comprising at most four fused rings, each ring has 3- 8 yuan and in each ring optionally comprising independently selected from O, S and N 1-4 hetero atom；

Wherein R^*And R^NH or C independently is when occurring every time_1-12Hydrocarbon, optionally by 1-6 selected from halogen, the miscellaneous original of O, S and N Son replaces；And two of which group R^*And/or R^N3-8 yuan of rings can together be formed.

13. is according to claim 1 for preparing bispecific antibody " AB " from first antibody " A " and SA " B " Or the method for " BA ", wherein the cycloaddition reaction occurs in the presence of copper ion.

14. is according to claim 1 for preparing bispecific antibody " AB " from first antibody " A " and SA " B " Or the method for " BA ", wherein the cycloaddition reaction occurs under neutral ph.

15. is according to claim 1 for preparing bispecific antibody " AB " from first antibody " A " and SA " B " Or the method for " BA ", the disulfide bond between the heavy chain and light chain of wherein at least 90% hinge area disulfide bond cracking after substantially Keep complete.

16. is according to claim 1 for preparing bispecific antibody " AB " from first antibody " A " and SA " B " Or the method for " BA ", wherein antibody A or antibody B is IgG1 immunoglobulin (Ig)s.

17. is according to claim 1 for preparing bispecific antibody " AB " from first antibody " A " and SA " B " Or the method for " BA ", wherein antibody A or antibody B is IgG4 immunoglobulin (Ig)s.

18. is a kind of for from IgG1, IgG2 or IgG4 antibody-like or its Fab2 fragment " A " and IgG1, IgG2 or IgG4 antibody-like Or the method that its fragment " B " produces the bispecific antibody " AB " or " BA " of chemistry locking, it includes：

A () reduction has hinge residues sequence (the EU index numbers selected from CPPC, CPSC, SPPC and SPSC：Residue 226- 229) first antibody " A " and with hinge residues sequence (the EU index numbers selected from CPPC, CPSC, SPPC and SPSC：It is residual Base 226-229) antibody " B " to form incomplete antibody A and incomplete antibody-B, wherein antibody A combines the first target and antibody B combines the Two targets, any interchain or intrachain disulfide bond that thus reducing condition is destroyed in the hinge area of antibody A and antibody B；

B () is by one or two Cys residue 226 or/and 229 of the first compound and the antibody hinge core sequence of incomplete antibody A (EU index numbers：Residue 226 or/and 229) connection, to form the incomplete antibody A of connection, wherein first compound has choosing From following structure：

M=N, C

M '=N, C

Z=I, Br, SPh

Wherein N₃It is-N=N=N；

C () is by second compound and have resisting for hinge residues sequence (residue 226-229) CPPC or CPSC or SPPC or SPSC One or two Cys residue 226 and 229 of the hinge core sequence of body B connects to form the antibody B of connection, wherein this second Compound has selected from following structure：

M=N, C

M '=N, C

Z=I, Br, SPh；

D () incubates in neutral conditions the antibody A and the antibody B being connected of the connection of approximately equal mole, to form chemical lock Fixed bispecific antibody AB.

19. methods for producing the bispecific antibody of chemistry locking according to claim 18, wherein the antibody A Reduction is carried out with forming incomplete antibody B with forming incomplete antibody A and antibody B reduction in reducing agent, and wherein the reducing agent is selected from L- Cysteine, dithiothreitol (DTT), beta -mercaptoethanol, cysteamine, TCEP (three (2- carboxyethyls) phosphines), 2-MEA (2-MEA) And combinations thereof.

20. methods for producing the bispecific antibody of chemistry locking according to claim 18, wherein with two Cys residues (EU index numbers：The hinge area of residue 226 or/and antibody A 229) is connected with part A, wherein part A tools Have selected from following structure：

M=N, C

M '=N, C

Z=I, Br, SPh

Wherein N₃It is-N=N=N.

21. methods for producing the bispecific antibody of chemistry locking according to claim 18, wherein with one Or two Cys residues (EU index numbers：The hinge area of residue 226 or/and antibody B 229) is selected from array structure with having Part B connection：

M=N, C

M '=N, C

Z=I, Br, SPh

To form the incomplete antibody A with the connection selected from following structure：

M=N, C

M '=N, C

Z=I, Br, SPh

Wherein N₃It is-N=N=N；

With the antibody B with the connection selected from following structure：

M=N, C

M '=N, C

Z=I, Br, SPh.

A kind of 22. bispecific antibody AB of chemistry locking, it includes the incomplete antibody A of connection, and it is connected to：

Wherein N₃It is-N=N=N；

The incomplete antibody A of the connection is incorporated into the antibody B of connection, and the antibody B of the connection is connected to：

Bispecific antibody " AB " or " BA " that 23. one kind are locked from the chemistry of IgG antibody-likes " A " and IgG antibody-likes " B ", It is included and the incomplete antibody A being connected selected from following structure：

M=N, C

M '=N, C

Z=I, Br, SPh

Wherein N₃It is-N=N=N；

Incomplete antibody A is incorporated into and the incomplete antibody B being connected selected from following structure：

M=N, C

M '=N, C

Z=I, Br, SPh.

A kind of 24. bispecific antibodies, it is included：

A () includes the single heavy chain and first antibody fragment A' of light chain from antibody A, wherein the single heavy chain has one Or multiple reactive mercapto groups；

B () includes the single heavy chain and SA fragment B' of light chain from antibody B, wherein the single heavy chain has one Or multiple reactive mercapto groups；

The 1,2,3- tri- that wherein described first and second antibody fragment is formed by the cycloaddition reaction via azide and alkynes Azoles is covalently attached, and the azide is connected to the reactive sulfydryl in the first antibody fragment, and the alkynes by joint Reactive sulfydryl in the SA fragment is connected to by joint.

25. bispecific antibodies according to claim 24, wherein fragment A' and B' are immune from IgG1 or IgG4 Globulin.

A kind of 26. antibody fragments with joint covalent bonding, the joint is included to be had and cycloaddition can occur with azide The C of-C ≡ C- the keys of reaction₈Ring.